The long-term goal of this project is to understand how transcription by RNA polymerase II (RNApII) is coupled to RNA processing and termination. Earlier funding periods of this project produced a model in which the C-terminal domain (CTD) of the RNApII subunit Rpb1 displays characteristic phosphorylation patterns at different stages of the transcription cycle to promote binding of the appropriate factors for co-transcriptional RNA processing. The fundamental knowledge generated by this project provides significant insight into how the CTD phosphorylation cycle affects medically important processes such as the stimulation of HIV transcription by the viral Tat protein and the pausing of RNApII at developmentally regulated genes in embryonic stem cells. This project is necessary to better understand both the enzymes that mediate the changes in CTD phosphorylation (kinases, phosphatases, etc.) as well as the proteins that recognize these patterns. In the next funding period, three specific aims will be being pursued. The first is to create a system for directly analyzing CTD phosphorylation sites by mass spectrometry, avoiding all the pitfalls and caveats associated with the monoclonal antibodies that have been used to date. A modified CTD will be engineered that adds several basic residues and non-consensus repeats so that mass spectrometry can be used to distinguish individual proximal and distal repeats. The in vivo phosphorylations on these CTD fragments will be analyzed in wild-type cells and mutants of various CTD modifying enzymes. Analysis of RNApII associated with specific CTD binding proteins (both in vivo and in vitro) will also be performed to determine their CTD binding specificities. In the second aim, a series of CTD mutants will be constructed that incorporate a non- native, photoreactive amino acid in vivo. In vivo crosslinking and analysis of associated proteins will show whether CTD-associated factors preferentially associate with proximal or distal repeats. In the third aim, we will analyze RNApII elongation complexes that are blocked at specific locations in genes. Elongation will be blocked by either a mutant recombinase that covalently links to nicked DNA but cannot excise, or by non- cleaving Cas9/CRISPR complexes. Stalled complexes will be characterized by ChIP for CTD modifications and associated proteins. A time course will be used to analyze the clearance of these blocked complexes, as well as whether their removal is stalled in cells mutated in various transcription termination or repair coupling factors. Together, these experiments will greatly advance our understanding of the events that occur during transcription elongation, both during unimpeded elongation and upon blockage by DNA damage.